It is known that the impedance measurements make it possible to detect extremely small variations in the properties and chemical composition of the electrodes used for performing the measurement as well as of the surface thereof and the medium between them. This has allowed registering phenomena such as the formation of an antibody-antigen complex on the electrode surface (Bataillard, P. et al., Anal. Chem, 1988, 60, 2374). The first systems were unable to detect minor compounds and even less so at trace level in the samples analyzed. To increase the sensitivity of the technique and also to miniaturize the devices, transducers based on interdigitated electrodes were developed (P. van Gerwen et al., Sens. Actua. B, 1998, 49, 73). The use of these types of electrodes as chemical sensors by means of impedimetric measurements has been addressed in patents using various conceptions (WO2004044570) and essentially involves the immobilization of the compound which plays the role of receptor on the surface of the electrode and putting it in contact with the sample to be analyzed. If the complementary compound exists, it will bind to the receptor modifying the nature of the surface layer of the transducer and modifying the impedance thereof. The magnitude of change is proportional to the amount of compound bound to the transducer which in turn depends on the amount thereof present in the sample. The correlation can be performed with the impedance value at one or several interrogation frequencies or by adjusting the response according to the frequency to an equivalent circuit and correlating the value of one or more components with the analyte concentration.
Despite the above improvements, the systems still did not have the required sensitivity for the detection and quantification of compounds at trace level and even less so when involving compounds with low molecular weight (less than 1000 Dalton). A major improvement in the design of the transducers was the development of the interdigitated electrodes with insulating barriers between the conductive elements with a height of the order of the separation of the electrodes disclosed in patent ES2307430. This transducer device allows detecting low-molecular-weight compounds with a similar sensitivity to that of ELISA-type assays that use the same set of immunoreagents.
Along with the precedents in impedimetric sensor technology the precedent in multi-analyte-type immunochemical analysis techniques must be added. In general the multi-analyte methods are carried out by separation within each elementary immunoassay system such that the multi-analyte system is no more than a system of n-assays for different n-analytes. This system does not exploit the extreme selectivity shown by the antibodies towards their substrates (similar to that of enzymes towards their own). An improvement to the conventional system and that somehow is reminiscent of how the immune system works is the use of a cocktail of antibodies that will recognize the different analytes, since there will be no cross-reactions between them. This strategy is addressed in the work on a multi-analyte ELISA assay for the detection of different families of antibiotics in milk (Adrian J. et al, Anal and Bioanal Chem, 2008, 391, 1703). Since it involved low-molecular-weight molecules, this required a competitive type assay where the suitable competitor for each analyte had been immobilized separately in the well plate, which allowed obtaining signals separately for each one despite using a mixture of antibodies on the sample. This work demonstrates the possibility of working with mixtures of antibodies without the nonspecific signal being greater than in individual assays and without interference between the responses for different analytes.